Schoonover J R, Palmer G
Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77251-1892.
Biochemistry. 1991 Jul 30;30(30):7541-50. doi: 10.1021/bi00244a025.
The ability to isolate preparations of cytochrome oxidase which are highly homogeneous has facilitated a study of the effects of various reagents on the purified enzyme. The addition of either sodium formate, formamide, formaldehyde, or sodium nitrite to enzyme which reacts in a single rapid kinetic phase with cyanide causes a blue-shift of 4-6 nm of the net (cytochrome a + cytochrome a3) Soret maximum. Only the derivative prepared by adding sodium formate demonstrates measurable intensity in the g' = 12 region of the low-temperature electron paramagnetic resonance (EPR) spectrum. This g' = 12 resonance is characteristic of cytochrome oxidase which has undergone a modification at the binuclear center and thereby reacts sluggishly with cyanide. As the site of cyanide binding in resting enzyme as been demonstrated to be CuB [Yoshikawa, S., & Caughey, W.S. (1990) J. Biol. Chem. 265, 7945-7958], it is proposed that formate can bind to CuB and the fast to slow transition is rationalized by using this proposal. The g' = 12 signal is also produced upon the addition of sodium formate to mitochondrial preparations, suggesting that the species responsible for this behavior may have possible physiological relevance. Physical properties of the formate derivative and data for other reagents reacted with the fast-reacting enzyme preparation are presented.
能够分离出高度均一的细胞色素氧化酶制剂，这有助于研究各种试剂对纯化酶的影响。向与氰化物在单一快速动力学阶段反应的酶中添加甲酸钠、甲酰胺、甲醛或亚硝酸钠，会使净（细胞色素a + 细胞色素a3）索雷特最大吸收峰发生4 - 6纳米的蓝移。只有添加甲酸钠制备的衍生物在低温电子顺磁共振（EPR）光谱的g' = 12区域表现出可测量的强度。这种g' = 12共振是细胞色素氧化酶的特征，该酶在双核中心发生了修饰，因此与氰化物反应缓慢。由于已证明静止酶中氰化物的结合位点是CuB [吉川，S.，& 考伊，W.S.（1990）《生物化学杂志》265，7945 - 7958]，有人提出甲酸盐可以与CuB结合，并利用这一观点解释了从快速到缓慢转变的合理性。向线粒体制剂中添加甲酸钠时也会产生g' = 12信号，这表明导致这种行为的物质可能具有潜在的生理相关性。本文介绍了甲酸盐衍生物的物理性质以及与快速反应酶制剂反应的其他试剂的数据。
